Ticks and Rickettsiae Associated with Wild Animals Sold in Bush Meat Markets in Cameroon

Ticks are obligate blood-sucking parasites of wild animals and transmit many zoonotic microorganisms that can spread to domesticated animals and then to humans. In Cameroon, little is known about tick diversity among wildlife, especially for animals which are hunted for human consumption. Therefore, this survey was undertaken to investigate tick and Rickettsia species diversity parasitizing the wild animals sold in bush meat markets in Cameroon. In total, 686 ticks were collected and identified to the species level based on morphology, and some were genetically analyzed using the 16S rRNA gene. Eighteen tick species belonging to five genera were identified: Amblyomma spp. (Amblyomma compressum, Amblyomma flavomaculatum, and Amblyomma variegatum), Haemaphysalis spp. (Haemaphysalis camicasi, Haemaphysalis houyi, Haemaphysalis leachi, and Haemaphysalis parmata), Hyalomma spp. (Hyalomma nitidum, Hyalomma rufipes, and Hyalomma truncatum), Ixodes spp. (Ixodes rasus and Ixodes moreli), and Rhipicephalus spp. (Rhipicephalus guilhoni, Rhipicephalus moucheti, Rhipicephalus muhsamae, Rhipicephalus microplus, Rhipicephalus camicasi, and Rhipicephalus linnaei). In terms of Rickettsia important for public health, two Rickettsia spp., namely Rickettsia aeschlimannii and Rickettsia africae, were detected in Hyalomma spp. and Amblyomma spp., respectively. Distinct tick–pathogen patterns were present for divergent sequences of R. africae associated with exclusively A. variegatum vectors (type strain) versus vectors comprising A. compressum, A. flavomaculatum, and A. variegatum. This suggests possible effects of vector species population dynamics on pathogen population circulation dynamics. Furthermore, Candidatus Rickettsia africaustralis was detected for the first time in Cameroon in I. rasus. This study highlights the high diversity of ticks among wildlife sold in bush meat markets in Cameroon.


Introduction
Ticks are obligate blood-sucking arthropods which parasitize every class of terrestrial vertebrate, including mammals, birds, reptiles, and even amphibians [1]. Ticks are primarily parasites of wild animals, with only~10% of species feeding on domestic animals [2,3]. During feeding on their hosts, they can cause various clinical manifestations including tissue injury, body paralysis, and anemia during massive infestations [4]. Many tick species are known reservoirs and vectors for a multitude of pathogens such as helminths, protozoa, bacteria, and viruses [3]. Wild animals play a significant role as reservoirs for many tickborne infections, which can be easily spread to domestic animals and then to humans via tick infestations [5]. The emergence and resurgence of several tick-borne diseases continues to pose increasing public health concerns and economic impacts [6][7][8][9][10]. Although the vectorial significance of selected tick species infesting domesticated animals and humans is well-established, there are still many unknowns with respect to genetic diversity, species delimitation, and host distribution, particularly in wildlife [5,11].
In Cameroon, 53 ixodid tick species are known from domestic and wild fauna [12][13][14][15]. The majority of studies have focused on ticks of importance for domestic animals and livestock [16][17][18][19]. The most comprehensive study dates back to 1958 [14]. Nevertheless, information on tick species that infest wild animals in Cameroon remains scant. This lack of knowledge is compounded by the rapid decline and/or extinction of many wild species in the region, with associated losses among their highly specialized obligate hematophagous tick parasites [20,21]. Amblyomma rhinocerotis, a tick specialized to feed on rhinoceros (Diceros bicornis), is already extinct in Cameroon due to growing anthropogenic impacts [22]. To conserve poorly known but highly threatened tick species, which may form the basis of future discoveries yet unknown, a greater understanding of their distribution, ecology, and biology is required [20].
Six ixodid genera, Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus, which contain 34 species between them, have been reported from wildlife fauna in Cameroon [14]. Previous studies were done exclusively based on morphology using museum or private collections [12][13][14][15]. However, morphology alone can prove insufficient to differentiate between closely related tick species, especially when the specimens are damaged, engorged, or at immature stages. Molecular data applied in a phylogenetic comparative framework using verifiable and taxonomically confirmed sequence data can serve to improve the taxonomic robustness of biodiversity surveys. The aim of this study was to identify ticks parasitizing wild animals sold in the bush meat markets in Cameroon using morphological and genetic methods and, in addition, the associated Rickettsia spp.

Study Area
The study areas were localized in three large agroecological zones of Cameroon: the Sudano-Sahelian zone (AEZ I), the High Guinea Savannah zone (AEZ II), and the Humid Forest zone with bimodal rainfall (AEZ V) ( Figure 1). A strong climatic gradient runs from the humid forest in the south up to the dry Sudano-Sahelian in the far northern region. The rainy season in the Guinea Savannah zone is from April to October, whereas in the Sudano-Sahelian zone, it is from June to September. Annual rainfall ranges from 1400 to 1700 mm in the Guinea Savannah zone and from 800 to 1400 mm in the Sudano-Sahelian zone. The Humid Forest zone with bimodal rainfall lies at altitudes ranging between 400 and 1000 m above sea level. This zone is characterized by a humid forest and savannah mosaic. The average temperature in this zone is about 26 • C, and the annual average rainfall is 2456.8 mm.
These regions were selected because of the presence of hunting, butchering, and consumption of wild meat practices. These areas are also considered to be hot spots for zoonotic disease outbreaks [23]. More than 900,000 reptiles, birds, and mammals are sold each year by or to the rural and urban populations, corresponding to around 12,000 tons of terrestrial vertebrates [24]. Mammals represent the largest proportion of hunted animals, followed by reptiles, birds, and amphibians [24].

Tick Sampling
A cross-sectional study was conducted between June 2020 to November 2021 in five bush meat markets in five localities, Kaele (10 • 6 00 N 14 • 27 00 E), Soramboum (7 •   The sampling areas (red circle) were located in the climate zones High Guinea Savannah, Sudano-Sahelian, and Humid Forest with bimodal rainfall.
Wild animals sold in the bush meat markets of the above-mentioned places were selected randomly and/or conveniently after explaining the aims of the study and upon agreement of the sellers. Most animals were dead and presented individually on a table or on the ground, together with other farming products. No financial incentive was given. Domestic animals are rarely seen in such places, except live chickens, which are kept apart from the bush meat. Examination of each animal was done at the market with as little disturbance as possible. Selected animals were thoroughly examined for attached ticks.
Total DNA was extracted using the MagNA Pure LC RNA/DNA Kit (Roche, Mannheim, Germany) in a MagNA Pure LC instrument (Roche) according to the manufacturer's Wild animals sold in the bush meat markets of the above-mentioned places were selected randomly and/or conveniently after explaining the aims of the study and upon agreement of the sellers. Most animals were dead and presented individually on a table or on the ground, together with other farming products. No financial incentive was given. Domestic animals are rarely seen in such places, except live chickens, which are kept apart from the bush meat. Examination of each animal was done at the market with as little disturbance as possible. Selected animals were thoroughly examined for attached ticks.
Total DNA was extracted using the MagNA Pure LC RNA/DNA Kit (Roche, Mannheim, Germany) in a MagNA Pure LC instrument (Roche) according to the manufacturer's instructions. DNA was extracted from individual ticks (if a single tick was found on a single host) or pools (2-10 ticks per pool, if ticks belonged to the same species and developmental stage and were collected from the same animal). The extracted total DNA was stored at −80 • C until PCR analysis. The 16S rRNA gene was amplified using polymerase chain reaction (PCR) protocols as described by Halos et al. [32].

Sequencing and Phylogenetic Analysis
All obtained 16S rRNA gene sequences for ticks from this study (GenBank accessions: ON318327-ON318387), as well as additional data from GenBank, were compiled into two datasets to represent Rhipicephalinae (Rhipicephalus and Hyalomma: 117 sequences) and non-Rhipicephalinae (Ixodes, Haemaphysalis, and Amblyomma: 97 sequences). Sequences from GenBank were chosen to encompass the range of Rhipicephalus, Hyalomma, Amblyomma, Haemaphysalis, and Ixodes species that occur in Afrotropical areas as well as closely related species to counterfactually confirm species identification based on morphology. The validity of species identification using sequences from GenBank follows from recent studies that include large-scale taxonomic investigations to verify species identity via phylogenetic analysis and correlated morphology [33][34][35][36][37]. The prevalence of misidentified tick species among sequence data in GenBank is a growing problem that can only be addressed by large-scale comparative taxonomic studies. Sequence data were aligned using MAFFT (Q-INS-i, 200PAM/k = 2; Gap opening penalty, 1.53) [38]. The optimal nucleotide substitution model was selected using BIC calculations in W-IQTREE [39] and was determined as TPM2+F+G4 (Rhipicephalinae) and K3Pu+F+I+G4 (non-Rhipicephalinae). Maximum likelihood analysis was performed in MEGA v7.0.14 [40] with 1000 bootstraps as well as with calculation of pairwise p-distances. Average p-distances between conspecific sequences from GenBank and collected samples were calculated to determine species identification validity according to the generally accepted threshold of 5% or greater sequence divergence between species [33,35,[41][42][43].
Rickettsia spp. detection was done using real-time PCR to amplify a fragment of the gltA gene for screening. In this first step, 114 Rickettsia spp. positive pools were detected. All positive pools with CT below 35 were further investigated by amplifying the 23S-5S intergenic space gene using a conventional PCR. Sequence data for Rickettsia 23S-5S (ON333673-ON333740) obtained from this study were compiled with reference data from Genbank and aligned using MAFFT (Q-INS-i, 200PAM/k = 2; Gap opening penalty, 1.53) [38]. The optimal nucleotide substitution model was selected using BIC calculations in W-IQTREE [39] and was determined as HKY+F. Maximum likelihood analysis was performed in MEGA v7.0.14 [40] with 1000 bootstraps.

Tick Species and Hosts
Associations of tick species with different hosts are presented in Table 2. Of the 18 tick species infesting 13 species of wild animal, only two species, namely A. compressum and Ha. camicasi, were common on eight and seven different host species, respectively. Most A. compressum (251/286) and A. flavomaculatum (17/39) were found on pangolins, whereas A. variegatum (99/126) was found on Nile monitors. Rhipicephalus muhsamae (9/10) was associated with rats, with only one nymph found on Nile monitors. Ten out of eighteen species were recorded from Nile monitors, whereas only one species, A. compressum, was recorded from golden cats (n = 2). Four-toed hedgehogs were infested with eight different tick species. Five species (Hy. nitidum, Hy. rufipes, I. moreli, Rh. linnaei, and Rh. camicasi) were recorded from a single host species each ( Table 2). Rhipicephalus microplus was recorded for the first time on four-toed hedgehogs (31/32) and Nile monitors (1/32).

Phylogenetic Analysis of the Partial 16S rRNA Gene
Phylogenetic trees are shown in Figures 2 and 3. Amblyomma flavomaculatum, Ha. camicasi, Ha. houyi, I. moreli, and Rh. moucheti were sequenced for the first time. Additionally, one fully engorged Hyalomma female that was difficult to identify morphologically was confirmed phylogenetically as Hy. truncatum. Six I. rasus, three Rh. guilhoni, and one Rh. muhsamae collected from our study areas were sequenced and phylogenetically confirmed as well.

Tick Species Distribution in the Three Agroecological Zones
The relative abundance and distribution of each tick species for each agroecological zone is described in Table 3.
In the Sudano-Sahelian zone, eight tick species were recorded: A. flavomaculatum, A. variegatum, Ha. houyi, Hy. rufipes, Hy. truncatum, Rh. microplus, Rh. muhsamae, and Rh. linnaei. Rhipicephalus microplus is reported for the first time in this region and host. In the High Guinea Savannah zone, six tick species (Ha. camicasi, Ha. houyi, Hy. nitidum, Rh. moucheti, Rh. guilhoni, and Rh. camicasi) were collected from seven hosts. Haemaphysalis species were associated with seven hosts. Rh. moucheti is reported for the first time in this region. In the Humid Forest zone with bimodal rainfall, seven tick species were collected: A. compressum, A. flavomaculatum, Ha. camicasi, Ha. parmata, Ha. leachi, I. rasus, and I. moreli. In three cases, tick species were found in two different agroecological zones. Amblyomma flavomaculatum was found in the Sudano-Sahelian and Humid Forest with bimodal rainfall zones, whereas Ha. camicasi was found in the High Guinea Savannah and Humid Forest with bimodal rainfall zones, and Ha. houyi was found in the Sudano-Sahelian and High Guinea Savannah zones.

Discussion
Limited studies focused on ticks parasitizing wild animals in Central Africa and their role as carriers of rickettsial bacteria. Traditional hunting and bush meat markets play a central role in contact between animals and humans and the spread of diseases from rural to urban populations. The selection of animals sold at such markets does not necessarily reflect natural abundances of the respective animal fauna. It is, however, useful for studying the human-vector-parasite interface and the diversity of tick, pathogen, and reservoir species involved. Due to the nature of bush markets, potential limitations include ticks leaving their hosts before examination or moving from one animal to another, though the prevalence of such events are generally rare and can be controlled for with large sample sizes. Additionally, vendors usually offer only one of any game species, further limiting the possibility of cross-contamination.

Tick Species and Hosts
In total, eighteen tick species belonging to Amblyomma, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus genera were collected from thirteen wild animal species.
Three Amblyomma species were recorded from wild animals, with A. compressum and A. flavomaculatum predominantly found on pangolins (noting the endangered status of pangolins and it being illegal to trade these animals in the markets in Cameroon since April, 2020), but also on Nile monitors and monkeys, whereas A. variegatum were found on Nile monitors and Four-toed hedgehogs in the Sudano-Sahelian zone. Amblyomma compressum is known as an exclusive ectoparasite of three African pangolin species [28]. However, in our study, A. compressum was collected from seven unexpected hosts, namely warthogs, Nile monitors, African brush-tailed porcupines, monkeys, African gold cats, African civets, and antelopes. Adults and immatures were collected from pangolins, whereas adults and nymphs were collected from African civets, monkeys, and warthogs, with only adults collected from the remaining hosts. This finding suggests that A. compressum is not very host-specific, and that cohabitation of pangolins with unexpected hosts can lead to infestations of the pangolin tick. Amblyomma flavomaculatum is known as a reptile tick, especially on Nile monitors and Savannah monitors (Varanus exanthematicus) [14,44], and it was previously reported from both hosts in Cameroon [14]. However, in the present study, A. flavomaculatum was collected from four hosts other than Nile monitors, namely, pangolins, four-toed hedgehogs, monkeys, and warthogs. It seems the pangolin is also an important host, as 17 specimens were collected from this host, while 16 specimens were collected from Nile monitors. Adults and two nymphs were collected from Nile monitors, while only adults were collected from unexpected hosts.
In the genus Rhipicephalus, six species were recorded on Nile monitor, four-toed hedgehog, African savanna hare, red flanked duiker, monkey, and rodent hosts. Rhipicephalus microplus, Rh. muhsamae, and Rh. linnaei were exclusively found in Sudano-Sahelian areas, whereas Rh. guilhoni, Rh. moucheti, and Rh. camicasi were found in High Guinea Savannah areas. Rhipicephalus microplus is currently expanding its geographical and host range, having been previously reported on cattle in Humid Forest and High Guinea Savannah zones, but in our study, it was found in the Sudano-Sahelian dry savannah and steppes on Nile monitors and four-toed hedgehogs for the first time. This is important to note, as Rhipicephalus are involved in the transmission of viral, bacterial, and protozoan agents to domestic and wild animals as well as humans.
From the genus Hyalomma, three species were recorded: Hy. rufipes, Hy. truncatum, and Hy. nitidum. Adults of Hy. rufipes and Hy. truncatum were found on four-toed hedgehogs, whereas immatures of Hy. truncatum and Hy. nitidum were found on Nile monitors and red flanked duikers, respectively. The genus is well-represented in Cameroon by five species: Hy. impeltatum, Hy. impressum, Hy. rufipes, Hy. truncatum, and Hy. nitidum, which are parasites of ruminants, birds, small mammals, and often humans [14]. They are involved in the transmission of R. aeschlimannii and Crimean-Congo hemorrhagic fever to domestic and wild animals as well as humans.
In this study, four species, Ha. camicasi, Ha. houyi, Ha. leachi, and Ha. parmata, were recorded. The paraphyly between Ha. leachi lineages between Chad and Cameroon is puzzling, but it may indicate an unknown divergence (species or population) or misidentification associated with sequence data. The Ha. leachi complex remains a taxonomic challenge to date [45].
Ticks of the genus Ixodes with seven species are poorly studied in Cameroon. In this highly specialized group of ticks, many immature stages are not even known. Two species were reported: I. rasus and I. moreli.

Phylogenetic Analysis of the Partial 16S rRNA Gene
Five tick species collected in the present study, A. flavomaculatum, Ha. camicasi, Ha. houyi, I. moreli, and Rh. moucheti, were sequenced for the first time. The phylogenetic analysis and sequences submitted to GenBank are relevant for future studies. Additionally, sequences confirmed morphological determinations for fully engorged specimens, larvae, and/or nymphs as well as for species collected from unexpected wild animal hosts like Rh. microplus and Rh. linneai. In total, 214 specimens were sequenced and included in the genetic analysis. Phylogenetic analysis confirmed the presence of Rh. camicasi in Cameroon, in sympatry with Rh. guilhoni. Both species belong to the Rhipicephalus sanguineus group and have a similar morphology, but they have different geographic distributions [29].

Rickettsia spp. Detection
The genus Amblyomma is involved in the transmission of viral, bacterial, and protozoan agents to domestic and wild animals as well as humans. These include Ehrlichia ruminantium (the agent of heartwater), Theileria mutans, and Rickettsia africae (the agent of African tick-bite fever) [46]. There are many reports about A. variegatum carrying R. africae from different African countries such as Sudan [47,48] and Ethiopia [49]. In the present study, R. africae was detected in A. compressum, A. variegatum, and A. flavomaculatum and was sequenced from 61 tests (92.4% of the 66 sequenced samples). Phylogenetic analysis showed three clusters of R. africae detected in A. variegatum but only one cluster for A. compressum (primarily), A. variegatum, and A. flavomaculatum. The mean sequence divergence between A. variegatumexclusive sequences and the wider vector associated clade (albeit primarily associated with A. compressum) was 0.006%. This may be related to the particulars of distinct tick species and their population dynamics, and it may not be directly linked with tick-host-pathogen biochemistry. More precisely, the high phylogenetic diversity of R. africae in A. variegatum may be due to the wide host range of A. variegatum that adds complexity and redundancy to the patterns of population dynamics among R. africae and A. variegatum populations. Furthermore, this suggests possible effects of vector species population dynamics on pathogen population circulation dynamics in the context of host population dynamics (a triple-layered system), which may drive evolutionary divergences in Rickettsia populations, strains, and species, or it may potentially cause differential pathogenicities to emerge over significant evolutionary time. Rickettsia aeschlimannii is associated with the Hyalomma species, especially with Hy. rufipes in Africa [47,48] as well as in European countries where Hy. rufipes is introduced by migratory birds as nymphs [50,51]. Rickettsia aeschlimannii was detected in two Hy. rufipes and one Hy. truncatum. The low number of R. aeschlimannii reported here is possibly due to the reduced number of Hyalomma species on wild animals.
Candidatus Rickettsia africaustralis was detected for the first time in Cameroon in two I. rasus females, both collected from pangolins. This Rickettsia species was detected for the first time in small mammal tissue from South Africa [52]. Ixodes rasus may prove to be a vector and reservoir for Candidatus R. africaustralis, as in other related Ixodes spp. Further investigations are necessary regarding the pathogenicity and vector competency for this new Rickettsia species.

Conclusions
The present study was carried out to determine the tick species that infest wild animals in Cameroon. As far as is known, this is the first tick survey of wild animals comprising tick species among five genera from well-defined hosts and agroecological zones. Ticks were identified morphologically and further confirmed or characterized genetically. In total, eighteen tick species belonging to Amblyomma, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus genera were collected from thirteen wild animal species. The most common bush meat species were the brush-tailed porcupine (Atherurus africanus), duiker (Cephalophus monticola), pangolin (Phataginus tricuspis), and Nile monitor (Varanus niloticus) [24]. Interactions between wild hosts, vectors, and pathogens are important to understand potential epidemiological risks and drivers.